Associations of body shape phenotypes with sex steroids and their binding proteins in the UK Biobank cohort

Associations of sex steroids and their binding proteins with body shape are unclear, because waist and hip circumference are correlated strongly with body size. We defined body shape using “a body shape index” (ABSI) and hip index (HI), which are independent of weight and height by design, and examined associations in multivariable generalised linear models for the UK Biobank cohort (179,902 men, 207,444 women). Total testosterone was associated inversely with ABSI, especially in men. Free testosterone was lowest for large-ABSI-large-HI (“wide”) and highest for small-ABSI-small-HI (“slim”) in men, but lowest for small-ABSI-large-HI (“pear”) and highest for large-ABSI-small-HI (“apple”) in women. Oestradiol was associated inversely with ABSI in obese pre-menopausal women but positively with HI in obese men and post-menopausal women not using hormone replacement therapy. Sex-hormone binding globulin (SHBG) was associated inversely with ABSI but positively with HI and was lowest for “apple” and highest for “pear” phenotype in both sexes. Albumin was associated inversely with HI in women, but matched the pattern of free testosterone in obese men (lowest for “wide”, highest for “slim” phenotype). In conclusion, sex steroids and their binding proteins are associated with body shape, including hip as well as waist size, independent of body size.


S4
Calculation of free sex steroid fractions Free testosterone and oestradiol were calculated according to the law-of-mass-action equations proposed by Sodergard et al. [ref. 16].
[S] (measured) and [fS] (calculated) were, correspondingly, the concentrations of the total and the free sex steroid of interest (testosterone or oestradiol) in mol/L. [SHBG] was the measured concentration of SHBG in mol/L. Conversion from nmol/L to mol/L involved a division by 10 9 . Conversion from mol/L involved a multiplication with 10 9 .
[albumin] was the measured concentration of albumin, converted from g/L to mol/L by division with the molecular weight 69,000 Da.

Definition of covariates
Age at enrolment (used on a continuous scale with 5 years increment), region of the assessment centre, weight change during the last year preceding enrolment, smoking status, physical activity, alcohol consumption, prevalent and incident cancers, and deaths (used for exclusions) were defined as previously described [ref. 3].
Fasting time was based on Field [74-0.0] "Fasting time". Three categories were defined as follows: 0-2 hours, [3][4] hours, 5 or more hours. Samples with unknown fasting time were assigned the sex-specific median category [3][4] hours. On a continuous scale, missing values were replaced with the sex-specific median (3 hours for both sexes).
Time of blood collection was based on Field [3166-0.0] "Time blood sample collected".

S5
Hand grip strength, used as an indicator of muscle functionality, was defined as in [ref. 11].
Missing values were replaced with the sex-specific median (26 for women, 42 for men).
Cholesterol lowering drugs use was based on Fields [6153-0.0/3] "Medication for cholesterol, blood pressure, diabetes, or take exogenous hormones", Question: "Do you regularly take any of the following medications? (You can select more than one answer)". Participants providing Answer 1 "Cholesterol lowering medication" were assigned to category Yes and those providing answer -7 "None of the above" or only answers 2 "Blood pressure medication", or 3 "Insulin", or in women 4 "Hormone replacement therapy", or 5 "Oral contraceptive pill or minipill" were assigned to category No. For the remaining participants, this information was considered missing and for them was used the sex-specific median category, No for both sexes.
Menopausal status (MP) was defined in three categories: Post-menopausal  We, therefore, excluded women with undetermined menopausal status and set age restrictions for subgroups by menopausal status (see the definitions of subgroups in women below).
Time of menstrual period was defined for pre-menopausal women and was based on Field [3700-0.0] "Time since last menstrual period"; Question: "How many days since your last menstrual period?". Answers -1 "Do not know" and -3 "Prefer not to answer" were considered missing values.
Subgroups of women were defined based on self-reported menopausal status, with restriction for age and use of oral contraceptives and HRT (see definitions of these variables above and Supplementary Figure S1 for illustration). Pre-MP -included pre-menopausal women younger than 55 years of age, not using oral contraceptives or cholesterol lowering drugs at enrolment and never using HRT. Only a small number of pre-menopausal women younger than 55 years and not using oral contraceptives at enrolment had used HRT in the past (n=650) or were using HRT at enrolment (n=684), which precluded examining these as separate subgroups. The number of premenopausal women never HRT users using cholesterol lowering drugs at enrolment was also low (n=862) and they were excluded from the subgroup; Post-MP Never-HRT -included postmenopausal women aged 50 years or older who have never used HRT and were not using oral contraceptives at enrolment; Post-MP Former-HRT -included post-menopausal women aged 50 S8 years or older who had used HRT in the past and were not using oral contraceptives at enrolment; Post-MP Current-HRT -included post-menopausal women aged 50 years or older who were using HRT but not oral contraceptives at enrolment. The transitional period (≥50 to <55 years) included 9,118 pre-menopausal women (18.2% of all pre-menopausal women) and 17,964 postmenopausal women (13.4% of all post-menopausal women). Women with self-reported premenopausal status but aged 55 years or older (n=1,330, 2.7 % of pre-menopausal women) and women with self-reported post-menopausal status but younger than 50 years (n=4,405, 3.3 % of post-menopausal women) were excluded from the subgroup analyses, as they were a small proportion and unrepresentative of the subgroup of women with the corresponding menopausal status.
Time since stopped HRT use (past HRT use) was derived for the study dataset by subtracting Field [3546-0.0] "Age last used hormone-replacement therapy (HRT)"; Question: "How old were you when you last used HRT?" from Age at enrolment. Answers -1 "Do not know" and -3 " Prefer not to answer" were considered missing values. To accommodate the large missingness, three categories were defined as follows: < 7 years (close to the median of the study dataset for women with past HRT use) (n=29,004 in the study dataset), ≥ 7 years (n=26,508), or Unknown (n=7,462).
Duration of HRT use (past HRT use) was derived for the study dataset by subtracting Field Answers -1 "Do not know" and -3 "Prefer not to answer" were considered missing values. To accommodate the large missingness, three categories were defined as follows: < 6 years (close to the median of the study dataset for women with past HRT use) (n=28,019 in the study dataset), ≥ 6 years (n=25,888), or Unknown duration (n=9,067).
Duration of HRT use (current HRT use) was derived for the study dataset by subtracting Field [3536-0.0] "Age started hormone-replacement therapy (HRT)" from Age at enrolment. To accommodate the large missingness, three categories were defined as follows: < 11 years (close to the median of the study dataset for women with current HRT use) (n=9,080 in the study dataset), ≥ 11 years (n=7,179), or Unknown duration (n=1,653).
HRT use and duration was used as covariate in models for women overall and was defined for the study dataset as a combined variable with seven categories as follows: Never use (n=126,558 in the study dataset); Past use <6 years (n=28,019); Past user ≥6 years (n=25,888); Past use Unknown duration (n=9,067); Current use <11 years (n=9,080); Current use ≥11 years (n=7,179); Current use Unknown duration (n=1,653).
Time since stopped oral contraceptives use (pre-menopausal, past use) was derived for the study dataset by subtracting Field [2804-0.0] "Age when last used oral contraceptive pill"; Question: "How old were you when you last used the contraceptive pill?" from Age at enrolment.
To accommodate the large missingness, four categories were defined as follows: < 10 years (n=8,382 in the study dataset), ≥ 10 to <20 years (n=16,232), ≥ 20 years (n=12,967), or Unknown S9 time (n=2,288). The cut-offs correspond to the tertile boundaries for pre-menopausal women with past OC use rounded to ten years.
Time since stopped oral contraceptives use (post-menopausal or undetermined, past use) was derived as for pre-menopausal women, but the four categories were defined as follows: < 20 years (n=21,594 for the study dataset), ≥ 20 to <30 years (n=42,037), ≥ 30 years (n=45,157), or Unknown time (n=14,334). The cut-offs correspond to the tertile boundaries for women with postmenopausal or undetermined status and past oral contraceptives, use rounded to ten years.
Oral contraceptives use with time since stopped was defined for the study dataset as a combined variable with six categories as follows: Never use (n=35,855 for the study dataset); Past use <10 (or<20) years (n=29,976); Past use ≥10 to <20 (or ≥20 to <30) years (n=58,269); Past use ≥20 (or ≥30) years (n=58,124), Past use Unknown time (n=16,632); Current use (n=8,588). The alternative cut-offs correspond to pre-menopausal women (or women with post-menopausal or undetermined status).
Age at menarche was based on Field [2714-0.0] "Age when periods started (menarche)"; Question: "How old were you when your periods started?". Answers -1 "Do not know" and -3 " Prefer not to answer" were considered missing values.
Age at menopause was defined for post-menopausal women and was based on Field Question: "How old were you when you had BOTH ovaries removed?", using the lesser of the two ages when both were available. Answers -1 "Do not know" and -3 "Prefer not to answer" were considered missing values. S10 Supplementary OC -oral contraceptives, cut-offs for duration of use correspond to pre-menopausal (post-menopausal or undetermined) menopausal status; SDstandard deviation; n (%) -number (percentage from total per column); # -number (percentage from Post-MP Current-HRT, overall).

Post-MP
SDdiff (95% CI) -estimates for standard deviation differences (95% confidence interval) were obtained from multivariable linear regression models with SHBG, albumin, total testosterone, free testosterone, or total or free oestradiol (Pre-MP) as an outcome variable; OR (95% CI) -estimates for odds ratios of oestradiol detection (95% confidence interval) were obtained from multivariable logistic regression models. All models included ABSI, HI and BMI on a continuous standard deviation scale (sex-specific z-scores), with adjustment for height, age at enrolment, weight change within the last year preceding enrolment, smoking status, alcohol consumption, physical activity, Townsend deprivation index, region of the assessment centre, time of blood collection, fasting time, use of cholesterol lowering drugs (except Pre-MP), and in women also age at the last live birth, oral contraceptives use with time since stopped, bilateral oophorectomy (except Pre-MP) and, additionally, menopausal status and HRT use and duration ( Covariates are defined in Supplementary Methods. * -p<0.05 from Wald test for the individual term; ** -p<0.0001.